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PubMed2002

The intracellular antibody capture technology (IACT): towards a consensus sequence for intracellular antibodies.

We describe the application of an intracellular antibody capture technology (IACT) as a generic in vivo selection procedure for isolating intracellular antibodies or ICAbs. IACT was applied to the de novo selection of functional ICAbs against the microtubule-associated protein TAU, found in neurofibrillary lesions of Alzheimer's disease brains. A panel of 17 different ICAbs was created which bind TAU inside cells and the epitopes recognized by the selected ICAbs have been determined by an in vivo epitope mapping procedure. Finally, sequence analysis showed that the IACT-derived ICAbs are characterized by a common signature of conserved amino acid residues, suggesting that the IACT naturally selects a sort of "captured consensus sequence" for intracellular antibodies. The development of IACT, together with the possibility of scaling up in a high throughput and automated format, makes IACT a new enabling tool for target validation in functional genomics and global proteomics.

Alzheimer Disease↗

PubMed2002

Intracellular antibody capture technology: application to selection of intracellular antibodies recognising the BCR-ABL oncogenic protein.

The expression of antibodies inside cells to ablate protein function has the potential for disease therapy and for target validation in functional genomics. However, due to inefficient expression or folding, only a few antibodies or antibody fragments, usually as single-chain Fv antibody fragments (scFv), bind their antigens in an intracellular environment. We have established a genetic-selection technology (intracellular antibody capture, IAC) to facilitate the isolation of functional intracellular scFv from a diverse repertoire. This approach comprises an in vitro library screen with scFv-expressing bacteriophage, employing bacterially expressed antigen, followed by a yeast in vivo antibody-antigen interaction screen of the sub-library of in vitro scFv antigen-binders. Accordingly, we have isolated panels of scFv that bind intracellularly to the BCR or the ABL parts of the BCR-ABL oncogenic protein. Sequence analysis of the intracellular antibody scFv panels revealed a sequence conservation indicating an intracellular antibody consensus for both VH and VL, which could form the basis for the de novo synthesis of intracellular antibody libraries to be used with intracellular antibody-capture technology.

Amino Acid Sequence↗

PubMed1998

Development of a PCR microplate-capture hybridization method for simple, fast and sensitive detection of Salmonella serovars in food.

The authors have developed an easy and rapid detection and identification system for Salmonella spp. in food. The gene inv A was selected as the target sequence. Oligonucleotides derived from conserved regions of this gene were able to exclusively prime the amplification of a 389 bp fragment when Salmonella spp. DNA was used as the template. An internal Salmonella spp. specific DNA probe was used for confirmation of the amplified polymerase chain reaction(PCR)product, by Southern blot or microplate-capture hybridization assay. In this fashion the sensitivity of the method was increased 100-fold (4.5 fg total DNA). To validate the method, a total of 75 food samples were tested. The PCR-microplate capture hybridization assay is easy to perform and much faster than traditional detection methods for Salmonella spp. in food. Hybridization in microtitre plates is more readily observed than in Southern blot and is more sensitive than conventional agarose gel electrophoresis.

Biotin↗

PubMed2002

Serogroup specific single and multiplex PCR with pre-enrichment culture and immuno-magnetic bead capture for identifying strains of D. nodosus in sheep with footrot prior to vaccination.

The identification of Dichelobacter nodosus present in a flock is a prerequisite to specific (autogenous) vaccination. Current methods of identification of the serogroup present in a population requires that the organisms be isolated, identified visually in mixed culture on streak plates, subcultured to purify and subjected to antigenic analysis. This process takes at least 3 to 4 weeks. This study describes the development of a simple and rapid serogroup specific PCR test for D. nodosus. A common forward primer was designed from the conserved amino-terminal region of the fimbrial gene (fimA) and 9 (A-I) serogroup specific reverse primers were designed from the carboxy-terminal regions of fimA of the different serogroups. To verify the specificity within D. nodosus, each specific primer pair was tested in PCR against 18 serogroups/serotypes (prototypes) and found to be specific for all the serotypes within the homologous serogroups. Eighty four other bacterial strains, either commonly occurring in sheep or found in the environment of sheep, and including organisms related taxonomically to D. nodosus, were used to check the specificity of these assays. They were found to be specific for D. nodosus as none of the 84 bacterial stains reacted. These primers detected 1 pg of purified chromosomal DNA, or 50-100 cells of D. nodosus in crude lysates. Sensitivity was markedly improved when an immuno-magnetic capture was employed. Single tube multiplex PCRs were tested with different combinations of common forward primer and groups of 3, 4 or 5 reverse primers chosen so that amplicon size for each reaction product was different. These were able to amplify DNA of isolates from all the relevant serogroups included in the reactions. These tests were evaluated with samples taken directly from lesions of footrot, either directly or preceded by DNA purification, immuno-magnetic capture, enrichment broth culture and culture on hoof agar media. Of these methods only PCR on mixed colonies from 4-day-old cultures on 4% hoof agar media yielded results of practical value.

Animals↗

PubMed1996

Policy Capturing with Ridge Regression

A policy capturing method combining human judgment with ridge regression is offered which results in superior judgment policy models. The new method (termed smart ridge regression) was tested against four others in seven judgment policy capturing applications. Performance criteria were two cross-validation indices: cross-validated multiple correlation and mean squared error of prediction of new judgments. Smart ridge regression was found to outperform ordinary least squares regression and conventional ridge regression, as well as subjective weighting and equal weighting of cues.

Journal Article↗

PubMed2001

Analysis of a Bacteroides conjugative transposon using a novel "targeted capture" model system.

Large conjugative transposons (CTn's) are widespread among Bacteroides spp. and they are responsible for the high rates of Bacteroides tetracycline resistance, which is mediated by the tetQ gene. These elements are self-transmissible and conjugation can be induced up to 1000-fold by the addition of tetracycline to cultures prior to mating. In addition to self-transfer, the Bacteroides CTn's, such as CTn341, are able to mobilize unlinked genetic elements such as plasmids and mobilizable transposons in a tetracycline-inducible manner. To study the molecular properties of these unique elements, a vector was designed to capture CTn's for analysis in heterologous hosts. This plasmid, pFD670, consisted of the low-copy vector pWSK29, the RK2 oriT, an ermF gene, and a tetQ gene fragment containing the N-terminus and promoter. The vector was transferred into Bacteroides recipients containing CTn341 where it integrated into the tetQ gene by homologous recombination. This integrated construct then was transferred back into an Escherichia coli host where it replicated as a plasmid, pFD699, about 56 kb in size. Further analysis showed that pFD699 could be transferred into Bacteroides hosts where it displayed the same tetracycline-inducible properties as the native CTn341. The captured element appeared to utilize a circular intermediate in both transfer and transposition, and integration into the chromosome seemed to be random. Hybridization studies with a range of Bacteroides CTn's encoding tetracycline resistance revealed a great deal of homology between most of the CTn's but there was much variation seen in the restriction patterns of these elements, suggesting great diversity among this group.

Bacteroides↗

PubMed1998

Murine antibodies against E2 and hypervariable region 1 cross-reactively capture hepatitis C virus.

The absence of readily available animal and cell culture models for hepatitis C virus (HCV) replication has bottlenecked research on protective immunity to HCV infection. Antibodies reactive with HCV virions in vitro are assumed to be candidates for neutralizing or inhibitory antibodies against HCV. To find potentially neutralizing or inhibitory antibody candidates, anti-C, anti-E1, anti-E2, and anti-HVR1 antisera acquired from mice immunized with corresponding recombinant proteins or synthetic peptides were used to capture HCV viral particles in vitro based on antibody-virus interaction assays. Both anti-E2 and anti-HVR1 antibodies effectively captured HCV in vitro. Furthermore, it was found that anti-E2 and anti-HVR1 antibodies could immunoprecipitate an isolate of HCV unrelated to the original antigenic HCV isolate. ELISA confirmed that anti-HVR1 antibodies cross-reactively bind to these unrelated HVR1 peptides. These findings suggest that anti-E2 and anti-HVR1 antibodies induced in mice have the ability to bind with HCV particles in an isolate cross-reactive manner and highlight the possible application of combining several sequences of HVR1 to generate broadly reactive anti-HVR1 antibodies.

Amino Acid Sequence↗

PubMed2000

Monoclonal antibodies to the hypervariable region 1 of hepatitis C virus capture virus and inhibit virus adsorption to susceptible cells in vitro.

To analyze the neutralizing-related activity of antibodies against the hypervariable region 1 (HVR1) of hepatitis C virus (HCV) in more detail, monoclonal antibodies (mAbs) against HVR1 were raised by immunizing various strains of mice with one of two synthetic HVR1 peptides that had been derived from two isolates of HCV. The epitope specificity of all six mAbs could be assigned by the use of a series of linear peptides in competitive ELISA. It seems that most subregions in the amino acid sequence of HVR1 can induce a humoral immune response in mice. All three mAbs specific to HVR1-6-1 had the ability to capture homologous HCV-6 and inhibit its absorption to susceptible cells in vitro despite the fact that the epitope of each mAb was at a different location in HVR1, whereas the other three mAbs specific to HVR1-7 could not capture HCV-6 nor inhibit the absorption of HCV-6 to susceptible cells. The data in this study suggest that mAbs against HVR1 can prevent the infectivity of HCV in an isolate-specific and epitope position-independent manner.

Adsorption↗

PubMed2025

Capturing Chromatin Organization by MNase-seq and ATAC-seq.

Hox genes play a pivotal role during development. Their expression is tightly controlled in a spatiotemporal manner, ensuring that specific body structures develop at the correct locations and times during development. Various genomics approaches have been used to capture temporal and dynamic regulation of Hox gene expression at the nucleosome/chromatin level. This chapter focuses on the utilization of capture MNase-seq and Assay for Transposase-Accessible Chromatin using sequencing (ATAC-seq), two advanced techniques that enable the exploration of chromatin accessibility and nucleosome positioning within these critical genomic regions.

Chromatin↗

PubMed1990

Neutron capture therapy beams at the MIT Research Reactor.

Several neutron beams that could be used for neutron capture therapy at MITR-II are dosimetrically characterized and their suitability for the treatment of glioblastoma multiforme and other types of tumors are described. The types of neutron beams studied are: 1) those filtered by various thicknesses of cadmium, D2O, 6Li, and bismuth; and 2) epithermal beams achieved by filtration with aluminum, sulfur, cadmium, 6Li, and bismuth. Measured dose vs. depth data are presented in polyethylene phantom with references to what can be expected in brain. The results indicate that both types of neutron beams are useful for neutron capture therapy. The first type of neutron beams have good therapeutic advantage depths (approximately 5 cm) and excellent in-phantom ratios of therapeutic dose to background dose. Such beams would be useful for treating tumors located at relatively shallow depths in the brain. On the other hand, the second type of neutron beams have superior therapeutic advantage depths (greater than 6 cm) and good in-phantom therapeutic advantage ratios. Such beams, when used along with bilateral irradiation schemes, would be able to treat tumors at any depth in the brain. Numerical examples of what could be achieved with these beams, using RBEs, fractionated-dose delivery, unilateral, and bilateral irradiation are presented in the paper. Finally, additional plans for further neutron beam development at MITR-II are discussed.

Biomedical Engineering↗

PubMed1988

Experience of boron-neutron capture therapy for malignant brain tumours--with special reference to the problems of postoperative CT follow-ups.

Boron-neutron capture therapy (BNCT) is theoretically a highly selective treatment of infiltrating tumours, in that the tumoricidal heavy particle radiation is limited to a sphere of 10 microns around a tumour cell which is loaded with non-radioactive boron-10 atoms. There were 73 gliomas among the 83 cases treated by boron-neutron capture therapy. For grade III-IV cerebral gliomas, 5 and 10 year survival rates were an unimpressive 19 and 10% respectively. This was the result of technical problems such as unsatisfactory reactors and inadequate craniotomies for the majority of the patients. If the analysis was limited to those whose tumours had been irradiated with more than 2.5 x 10(12) neutrons/cm2 (yielding more than 3,000 rem or more), the 5 and 10 year survival were almost 100 and 50%. The longest surviving glioblastoma (grade IV) patient has lived in a satisfactory manner for the past 15 years. For the cases who had been treated with borderline doses (lethal or sublethal), interpretation of the postoperative CTs was frequently intriguing. Several cases had to undergo re-opening and occasionally even another BNCT, only to find no viable tumour tissue. Death occurred in some, either due to discontinuation of supportive treatments by local physicians, or due to excessive therapies by the author directly involved in the patient's care, both of whom had erroneously believed in recurrence. At autopsy, residual tumour cells were recognized only in the areas where the above-mentioned neutron fluence had not been delivered at the time of the treatment.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗

PubMed1994

Capturing of host DNA by a plant retroelement: Bs1 encodes plasma membrane H(+)-ATPase domains.

The recently identified maize retroelement Bs1 encodes domains of the plasma membrane H(+)-ATPase. This is the first example of host DNA captured by a plant retroelement and resembles the acquisition of oncogenes by vertebrate retroviruses. The ability to capture sequences from its host provides plant retroelements with a mechanism to alter gene structure which could be important for evolutionary adaptive change.

Amino Acid Sequence↗

PubMed1992

Monoclonal antibody based capture ELISA/ELIFA for detection of Coxiella burnetii in clinical specimens.

A CAPTURE ELISA/ELIFA system based on monoclonal capture and biotinylated monoclonal detection antibody is described. The assay is fast, highly specific and detects a minimum dose of 2500 Coxiella (C.) burnetii particles. In contrast to the sophisticated and cumbersome isolation procedures, even non-specialized laboratories could use this assay system for investigating clinical samples of different origin for C. burnetii within a short period of time.

Antibodies, Monoclonal↗

PubMed1993

Cerebral neurons underlying prey capture movements in the pteropod mollusc, Clione limacina. I. Physiology, morphology.

The pteropod mollusc Clione limacina feeds on shelled pteropods capturing them with 3 pairs of oral appendages, called buccal cones. A group of electrically-coupled putative motoneurons (A neurons) has been identified in the cerebral ganglia, whose activation induces opening of the oral skin folds and extrusion of the buccal cones. These cells are normally silent and have one or two axons in the ipsilateral head nerves. Electrical coupling between A neurons is relatively weak and normally does not produce 1:1 spike synchronization. Coupling coefficients ranged from 0.05 to 0.25. A second type of putative motoneurons (B neurons) controls retraction and withdrawal of buccal cones. B neurons show spontaneous spike activity which maintains the buccal cones in a continuous retracted state. All B neurons have one axon running into the head nerves. Ipsilateral B motoneurons are electrically coupled to each other. A neurons strongly inhibit B neurons, however, seven identified A motoneurons which were specifically tested do not form monosynaptic contacts with B motoneurons. Appropriate stimuli from the prey activate A motoneurons, which in turn inhibit B motoneurons and evoke extrusion of the buccal cones. One mechanism promoting the speed of this extremely rapid reaction is brief co-activation of antagonistic A and B neuron groups, which provides a notable increase in fluid pressure inside the head. Mechanical stimulation of buccal cones provides excitatory inputs to A motoneurons. Similar stimulation from captured prey would serve to prolong buccal cone protraction during the manipulatory phase of feeding.

Animals↗

PubMed1978

Enzyme layers on glass as a new model for the quantitative study of capture reactions in cytochemistry, with special attention to acid phosphatase.

A model system is described for the study of capture reactions for diffusable compounds in enzyme cytochemistry. The model, which allows the investigation of the influence of the composition of the cytochemical medium, the enzymatic activity, and the dimensions of the enzymatic site on the capture reaction, consists of very thin homogeneous layers of enzyme (0.01-0.1 micrometer thick) on glass, which are incubated in the cytochemical medium. The fraction of the total amount of liberated product precipitated in the enzyme layer is dependent not only on the trapping efficiency of the cytochemical medium but also on the concentration of the primary reaction product that can be built up in the enzyme layer. Calculations were performed to determine the steady-state concentration of the primary reaction product that can be built up in the enzyme layer. Acid phosphatase was used as enzyme. The problems associated with the model and its applicability to other types of cytochemical reactions are discussed.

Acid Phosphatase↗

PubMed1987

Digital image capture and analysis for split-drop micropuncture.

A method for automated image capture and analysis of shrinking drop sequences is described. The procedure can be managed by a single operator and allows estimates of proximal tubule volume flux to be calculated during the experiment. Speed of analysis is increased considerably (mean time 1.5 min per sequence) compared with previous methods. The potential exists for measurement of droplets in tubules that follow a tortuous path in the horizontal plane and further increases in capture and analysis rate would allow estimation of volume flux under non-steady state conditions. Film processing is eliminated and the removal of operator errors, bias and fatigue associated with manual measurement, coupled with the greatly reduced time required for analysis of sequences makes the shrinking drop method much more reliable and attractive.

Animals↗

PubMed1983

Studies on the phenazine methosulphate-tetrazolium salt capture reaction in NAD(P)+-dependent dehydrogenase cytochemistry. I. Localization artefacts caused by the escape of reduced co-enzyme during cytochemical reactions for NAD(P)+-dependent dehydrogenases.

The correct localization of oxidative enzymes using cytochemical tetrazolium methods, in which low molecular weight electron carriers such as NAD(P)H and reduced phenazine methosulphate (PMSH) are used, can be endangered by the escape of the reduced intermediates before they react to form the insoluble formazan at the true enzyme-containing sites. To investigate this phenomenon, the glucose-6-phosphate dehydrogenase reaction was studied in fixed erythrocytes which, because of their microscopic dimensions, are well-suited for studying the loss of intermediates. A mixture of active and heat-inactivated fixed erythrocytes was incubated in a PMS-supplemented medium for glucose-6-phosphate dehydrogenase. The cytophotometric histograms showed that the final formazan precipitate was equally distributed over both active and inactivated cells. When bovine serum albumin was added to the medium, all the formazan was found to be bound to this protein and the erythrocytes remained essentially unstained. The false localization in this system could be explained by an unfavourable balance between the capture of electrons carried by NADPH within the erythrocyte and the diffusion of NADPH out of the erythrocyte. The rate constant of NADPH oxidation was determined, as was also the diffusion constant of NADPH in a protein matrix. Substituting the data obtained into formulae derived from the enzyme cytochemical localization theory of Holt & O'Sullivan (1958), it was calculated that the capture reaction was highly deficient and, theoretically, less than 1% of the total amount of formazan produced was localized within the erythrocyte which explains the false localization observed. The importance of these findings for the cytochemical demonstration of NAD(P)+-dependent dehydrogenases in cells and electropherograms is briefly discussed.

Coenzymes↗

PubMed1986

Pinocytic capture and exocytosis of rat immunoglobulin IgG-N-(2-hydroxypropyl)methacrylamide copolymer conjugates by rat visceral yolk sacs cultured in vitro.

Rat immunoglobulin (IgG) was covalently bound to N-(2-hydroxypropyl)methacrylamide (HPMA) copolymers via glycylglycyl spacer. The resultant conjugate, free IgG and HPMA copolymer (containing a low percentage of tyrosinamide to facilitate radiolabelling) were radioiodinated, and their rates of pinocytic uptake, intracellular degradation and exocytic release by rat visceral yolk sacs cultured in vitro were determined. Free IgG was pinocytosed rapidly by the yolk sac and some IgG was subject to intracellular proteolysis. In comparison the IgG-HPMA copolymer conjugate was captured more slowly, but faster than unmodified HPMA. IgG was also exocytosed rapidly by the yolk sac following pinocytic capture and similarly IgG-HPMA copolymer had a much higher rate of release than unmodified HPMA. Measurement of tissue accumulation of 125I-labelled IgG-HPMA copolymer in the presence of increasing concentrations of non-radiolabelled IgG showed competition for membrane binding sites between the free, and polymer-bound immunoglobulin. These experiments indicate that immunoglobulins can be covalently bound to a soluble polymer developed as a drug-carrier in such a way that they can still interact with specific membrane receptors and they are subsequently subjected to specific cellular transport mechanisms.

Animals↗